The invention relates to methods of identifying compounds that modulate mTORC1 activity in a cell by modulating the activity of SLC38A9 (NCBI Gene ID: 153129), as well as to the use of such identified compounds in the modulation of mTORC1 and the treatment of diseases and conditions characterized by aberrant mTORC1 activity.
The mammalian (mechanistic) target of rapamycin (mTOR) is a master regulator of cell, organ and organismal growth in response to nutrients, growth factors and stress factors. mTOR is a serine/threonine kinase and nucleates at least two distinct complexes, mTOR complex 1, mTORC1, and mTOR complex 2, mTORC2. mTORC1 activity is regulated in part by amino acids. When activated, mTORC1 can stimulate cell growth by promoting anabolic processes such as mRNA translation and inhibiting catabolic processes such as autophagy through the actions of its downstream substrates. However, aberrant mTORC1 activation has been observed in a wide range of diseases including multiple types of cancer, metabolic dysfunction (e.g. type 2 diabetes, obesity), auto-immune diseases (e.g. psoriasis) and neurodegenerative diseases (e.g. Alzheimer's and Parkinson's disease), neuropsychiatric syndromes (e.g. autism and major depressive disorders), skeletal muscle dysfunction (e.g. sarcopenia, disease induced cachexia and disuse atrophy), as well as several rare diseases including those resulting from inherited and acquired mitochondrial dysfunction (e.g. Leigh Syndrome) and certain inherited growth defects.
The mechanism by which amino acids regulate mTORC1 signaling is complex and is centered on the lysosome. In amino acid replete conditions, amino acids cross the plasma membrane and accumulate within the lumen of the lysosome. In addition, the degradation of protein in the lysosomal lumen by lysosomal proteases and peptidases can also contribute to the accumulation of amino acids within the lysosome and lead to mTORC1 activation. mTORC1 is recruited to the lysosomal membrane where it interacts with the Rag GTPases—obligate heterodimers of RagA or RagB with RagC or RagD. The Rag GTPase heterodimer does not contain lipid anchors tethering the complex to the lysosome. Instead, it relies upon the pentameric Ragulator complex, which is tethered to the lysosomal membrane via lipidation of the Ragulator component LAMTOR1 (p18) for lysosomal localization. Furthermore, the Ragulator components LAMTOR2-5 (p14, MP1, C7orf59 and HBXIP, respectively) assembled with LAMTOR1 acts as a guanine nucleotide exchange factor for RagA and RagB, fostering their loading with GTP. As a result, Ragulator is not only necessary for localizing the Rag GTPase heterodimer to the lysosomal surface, but for also catalyzing the activated nucleotide binding state of the RagA/B. Additional protein complexes responsive to the availability of amino acids have been identified that regulate the activity of the Ragulator and Rag complexes, but the precise mechanism by which individual amino acids are physically sensed by the mTORC1 pathway has not been elucidated.
Given the known role that leucine and arginine play in activating mTORC1, we hypothesized that proteins exist that are able to specifically sense leucine or arginine at the lysosome and in response activate the lysosomal machinery upstream of mTORC1. We sought to identify such proteins through proteomic and biochemical approaches.
Given the importance of mTORC1 modulation in both anabolic processes and in certain disease states, there is a need to identify other members of the mTORC1 activation pathway as potential targets for modulation, which in turn can modulate mTORC1 activity.